Understanding the relationship between bacteriophages and pathogenicity in the gut pathogen Clostridium difficile

Clostridium difficile associated diarrhoea (CDAD) is one of the most worrying emerging bacterial pathogens and over 2000 people in the UK were killed last year (Kuijper, 2006). Only two antibiotics can be used to treat C. difficile and resistance has developed towards them both. However, bacteria have viral enemies, or bacteriophages which specifically target them. All bacteria have bacteriophages and they have been engaged in an arms race for as long as they have existed. Bacteriophages represent a natural, specific and extensive resource of novel antimicrobial agents. They could be either used directly as therapeutic agents, or products derived from their genomes (e.g. lysins which degrade bacterial walls) could be harnessed to lyse them (Fischetti, 2004). Bacteriophages are used extensively to control other bacterial pathogens, they are used widely in Eastern Block countries, and in USA last year the FDA recently released approval to use them in hot dogs to control Listeria.

Whilst other bacterial pathogens have well characterised bacteriophages, those which infect C. difficile are very poorly known. This is in part due to the difficulties in working with the appropriately named C. difficile. To understand how bacteriophages could be used to control C. difficile first a comprehensive collection of them has to be isolated and characterised and an appropriate selection of them sequenced to characterise what components of their genomes could be exploited to control C. difficile.

A different set of bacteriophages integrate into the genomes of C. difficile and alter their virulence. Whilst hiding in the bacterial genomes, they can encode a range of toxins which increase their ability to proliferate in the gut and hospital environment. An understanding of the way in which bacteriophage achieve this can be related to hospital practices and may result in strategies to minimise the spread of virulence factors.

I will provide the first comparative study of the bacteriophages that are associated with patients who have C. difficile associated diarrhoea and compare them to those who have C. difficile but not diarrhoea. I will characterise both the lytic phages and those that reside in the C. difficile genomes. I will establish if diarrhoea free patients have a more effective natural bacteriophage population than those who have diarrhoea. Sequence information will allow me to characterise how bacteriophages kill C. difficile. It will also allow tests to be developed that can see how environmental stimuli can trigger toxin release in the ?hidden? bacteriophages.

Clostridium difficile associated diarrhoea (CDAD) is a major problem in UK hospitals and was responsible for more than 2000 deaths across the UK last year. It is thought to cost the NHS more money than any other bacterial pathogen. Only two antibiotics are available to treat C. difficile and resistance is developing to both of them. Like all bacteria, C. difficile is infected by a wide range of viruses (bacteriophages) which specifically target it. Bacteriophages are engaged in an arms race with their hosts and their genomes offer a rich source of novel antimicrobials. Lytic bacteriophages control the population structure and evolution of bacteria by selectively killing certain strains. The lysins and holins used for this purpose have enormous therapeutic potential. A different subset of bacteriophages integrate into C. difficile host genomes where they encode toxins or virulence factors which influence host pathogenicity. The bacteriophage community associated with C. difficile is poorly characterised and it is not known if specific phages infect either CDAD associated or non-CDAD associated C. difficile strains. My preliminary studies have shown that there is a diverse bacteriophage community associated with just seven C. difficile strains isolated from patients who had tested positive for the clostridial toxin tcdA. I propose to take an ecological approach to understanding the role of bacteriophages in CDAD. To determine their prevalence and diversity in C. difficile, I will isolate them from C. difficile colonised patients at the Leicester Royal Infirmary over a one year time period. I will characterise them according to their morphology, genome size and ecological attributes. These studies will direct the sequencing of 12 bacteriophage genomes, 6 associated with patients with CDAD, and 6 from C. difficile colonised but non-CDAD presenting patients. This will reveal novel lysins and holins and other pathogenicity factors. Genome information will be used to design molecular probes which will establish the genetic diversity of the C. difficile bacteriophage community. Differences in the composition of bacteriophage genomes can then be related to hospital practices and other environmental factors. A collection of well characterised bacteriophages will provide an essential resource which will underpin future biochemical and genetic analysis of novel therapeutic agents and establish how bacteriophages influence C. difficile virulence.